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Increasing temperature and varying light quality can favor the formation of blooms of toxin-producing cyanobacteria. With respect to light quality, increasing type-B ultraviolet radiation (UV-B) causes cellular damage with cascading effects at different levels of biological systems. This study aimed to evaluate the combined effects of light intensity, UV-B, and increasing temperature on the circadian rhythm of production and release of microcystins (MCs) as well as the expression of mcy genes (mcyA, mcyD, and mcyE) and internal H2O2 formation of Microcystis aeruginosa. Microcystis aeruginosa was exposed to UV-B (0.8 W m−2) in 24-h cycles (12 h light:12 h dark) at 23, 25, and 30 °C combined with 30 or 50 µmol photons m−2 s−1. Samples were collected every 4 h during each 24-h cycle for MC analyses, evaluation of mcy gene expression by RT-qPCR, and internal H2O2 formation. The combination of 23 and 30 °C with UV-B changed the circadian rhythm of MC production and release and reduced the cyanotoxin concentration per cell quota. mcyD gene expression was 10.75-fold higher at 23 °C and 50 µmol photons m−2 s−1 treatment. However, a 7.55-fold decline in the expression of the mcyE gene occurred at 12 h (light period). The combination of 23 °C with UV-B caused the highest intracellular H2O2 formation. The enhanced expression of genes involved in MC synthesis at midday demonstrated that there is a critical period of stress that signals the toxin-production, which has significant implications for the general well-being of aquatic ecosystems.

Endophytic fungi are microorganisms that colonize the interior of plant tissues (e.g. leaves, seeds, stem, trunk, roots, fruits, flowers) in intracellular and/or extracellular spaces without causing symptoms of disease in host plants. These microorganisms have been isolated from plant species in a wide variety of habitats worldwide, and it is estimated that all terrestrial plants are colonized by one or more species of endophytic fungus. In addition, these microorganisms have been drawing the attention of researchers because of their ability to synthesize a wide range of bioactive molecules with potential for applications in agriculture, medicine and biotechnology. However, several obstacles come up when studying the diversity and chemical potential of endophytic fungi. For example, the usage of an inappropriate surface disinfection method for plant tissue may not eliminate the epiphytic microbiota or may end up interfering with the endophytic mycobiota, which consequently generates erroneous results. Moreover, the composition of the culture medium and the culture conditions can favor the growth of certain species and inhibit others, which generates underestimated results. Other inconsistencies can arise from the fungus misidentification and consequent exploration of its chemical potential. Based on the methodological biases that may occur at all stages of studies dealing with endophytic fungi, the objective of this review is to discuss the main methods employed in these studies as well as highlight the challenges derived from the different approaches. We also report associated tips to help future studies on endophytic fungi as a contribution.

Multifaceted microorganisms such as the bacterium

Lavasan Lake is one of the main water sources for Tehran metropolitan region, besides irrigating 30,000 ha of agricultural land. Cases of animal deaths linked to water ingestion prompted us to study the occurrence of cyanobacteria-related toxic compounds. We investigated the production of microcystins (hepatotoxins) and homoanatoxin-a (neurotoxin) by isolated heterocyst‐forming cyanobacteria using LC-MS and MALDI-TOF mass spectrometry coupled with the detection of biosynthetic gene cluster sequences. The isolates were identified previously by combining morphological analysis and the 16S rRNA gene sequencing. Homoanatoxin-a was detected by LC-MS run at a retention time of 16.3 min, and the protonated molecular ion (m/z 180) was trapped and fragmented by mass spectrometry. The presence of the toxin-producing anaC gene was found in only one Anabaena isolate in agreement with the toxin homoanatoxin-a production. This is the first report of a neurotoxin-producing cyanobacterium isolated from Lavasan Lake.

Ajigol wetland is a complex ecosystem in Northern Iran, which is ecologically important because contains up to 50% of threatened native plant species and aquatic organisms. Increasing of fish death and animal poisonings has led to an increased research interest and public awareness of harmful cyanobacterial blooms in this area. In the present work, we characterized a filamentous heterocyst-forming cyanobacterium morphologically and molecularly. Coupled with the 16S rRNA gene sequence, the rbcL gene and the 16S–23S internal transcribed spacer (ITS) secondary structures were analysed. Further, sequences of the mcyD and mcyG genes of microcystin synthetase (mcy) cluster were PCR amplified and sequenced. The secondary structures of the D1-D1’, BOX-B and V3 regions provided a better resolution of the studied strain. The sequenced mcy genes involved in the microcystin synthesis contribute to extend the understanding of the evolution of mcy genes in the genus Fischerella.

Microcystins (MCs) are the most studied toxins of cyanobacteria in freshwater bodies worldwide. However, they are poorly documented in coastal waters in several parts of the world. In this study, we investigated the composition of cyanobacteria and the presence of microcystins (MCs) in several coastal aquatic ecosystems of Nigeria. Direct morphological analysis revealed that members of the genus Oscillatoria were dominant with five species, followed by Trichodesmium with two species in Nigerian coastal waters. Oso Ibanilo had the highest cyanobacterial biomass (998 × 103 cells/L), followed by Rivers Ocean (156 × 103 cells/L). Except for the Cross River Ocean, cyanobacteria were present in all the investigated aquatic ecosystems. Ten (10) out of twenty water bodies examined had detectable levels of MCs. Furthermore, genomic DNA analysis for the mcyE gene of microcystin synthetase (mcy) cluster showed identities higher than 86% (query coverage > 96%) with toxic strains of cyanobacteria in all the samples analyzed. Also, the sequences of samples matched those of uncultured cyanobacteria from recreational lakes in Southern Germany. Our findings indicate that the presence of toxic cyanobacteria in coastal waters of Nigeria is of public and environmental health concern.

Made available in DSpace on 2022-04-28T19:48:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Sphaerospermopsis aphanizomenoides is a filamentous nitrogen-fixing and bloom-forming cyanobacterium, which biomass can fertilize natural water with nutrients, especially through nitrogen fixation. The Sphaerospermopsis aphanizomenoides strain BCCUSP55 was previously isolated from a water supply reservoir in the Brazilian semiarid region, and its draft genome assembly coupled with the gene contents are reported here. The obtained BCCUSP55 draft genome comprised 254 scaffolds with a genome size estimated of 6,096,273 bp. In addition, it has 5250 predicted coding sequences (CDS) and the G + C content is 38.5%. Further, the BCCUSP55 draft genome presented the putative nocuolin A gene complete cluster, a natural oxadiazine that triggers apoptosis in human cancer cells. Thus, our results contribute to extend the knowledge on the genus Sphaerospermopsis and reveal its biotechnological potential. Department of Cellular Biology Institute of Biological Sciences University of Brasília (UnB) Department of Biological Sciences “Luiz de Queiroz” College of Agriculture University of São Paulo (USP) Department of Genetics “Luiz de Queiroz” College of Agriculture University of São Paulo (USP) School of Agricultural and Veterinarian Sciences São Paulo State University School of Agricultural and Veterinarian Sciences São Paulo State University CNPq: CNPq—442083/2014-9 FAPESP: FAPESP—2013/15296-2

Human and veterinary pharmaceuticals either in the form of un-metabolized, incompletely metabolized, and metabolized drugs are increasingly present in aquatic ecosystems. These active pharmaceutical ingredients from pharmaceutical industries, hospitals, agricultural, and domestic discharges find their way into water systems – where they adversely affect non-target organisms like phytoplankton. Different aspects of phytoplankton life; ranging from growth, reproduction, morphology, physiology, biochemical composition, oxidative response, proteomics, and transcriptomics are altered by pharmaceuticals. This review discusses the currently available information on the susceptibility of phytoplankton to the ever-increasing presence of pharmaceutical products in the aquatic environment by focusing on the effect of APIs on the physiology, metabolome, and proteome profiles of phytoplankton. We also highlight gaps in literature concerning the salient underlining biochemical interactions between phytoplankton communities and pharmaceuticals that require an in-depth investigation. This is all in a bid to understand the imminent dangers of the contamination of water bodies with pharmaceutical products and how this process unfavorably affects aquatic food webs.

The general overview aimed to increase the current knowledge interactions between dehydrogenase enzymes and nanoparticles, and introduce dehydrogenases for industrial and health purposes. Nanoparticles (NPs) are particles constituting from 1 to 100 nm based on their size with a surrounding interfacial layer. Nanoparticle-Protein interactions include covalent and non-covalent attachments. Several dehydrogenase enzymes (e.g., alcohol dehydrogenase, lactate dehydrogenase, alanine dehydrogenase, glutamate dehydrogenase, leucine dehydrogenase, phenylalanine dehydrogenase, and malate dehydrogenase) are used for immobilization by nanoparticles. Such as magnetic nanoparticles and quantum dots, represent attractive model systems for biological enzyme assemblies and design of bioanalytical sensors. Further, bioconjugation of nanoparticles with dehydrogenase enzymes has broad applications in biocatalysis and nanomedicine for drug discovery. However, studies on the characterization of nanoparticle-enzyme complexes accept apparent that the anatomy and action of enzymes are afflicted by the chemistry of nanoparticle ligand, size, actual, and labeling methods. Moreover, the nanoparticle-protein conjugation revealed increased/decreased enzymatic activity due to nanoparticle features. Thus, this work reviewed the findings of nanoparticle-enzyme interactions for nanotechnology applications and conjugation techniques. We also highlight several challenges associated with the nanoparticle-enzyme interactions, including stability and reusability of the enzymes in nanoparticle-enzyme formation.

Se realizó la caracterización de ficoeritrina de la cepa A5 de Nostoc sp., seguida de investigación de su actividad biológica para aplicaciones biotecnológicas. Para la extracción de ficoeritrina, el uso de tampón acetato (pH 5.1) produjo 65.04 µg mL-1, y se identificó como C-ficoeritrina. Los resultados de su actividad antioxidante sugirieron su acción como un potente eliminador de radicales libres. Además, la C-ficoeritrina de Nostoc mostró una capacidad notable como agente antibacteriano y antifúngico, con estabilidad significativa de hasta 10 días. La glucosa (4 mg mL-1) fue un buen conservante para la C-ficoeritrina a 25 y 4 ºC. Se obtuvo por primera vez una C-ficoeritrina estable de Nostoc sp. en condiciones de sequía en piedra caliza, lo que demuestra la necesidad de estudiar microorganismos de ambientes extremos. The phycoerythrin characterization from Nostoc sp. strain A5 was done, followed by investigation of its biological activity for biotechnological applications. For phycoerythrin extraction, the use of acetate buffer (pH 5.1) resulted in 65.04 µg mL-1, and C-phycoerythrin was identified. Results of its antioxidant activity suggested action as a potent free radical scavenger. In addition, Nostoc's C-phycoerythrin showed noteworthy ability for antibacterial and antifungal agents with significant stability up to 10 days. Glucose (4 mg mL-1) was a good preservative for C-phycoerythrin at 25 and 4 ºC. A stable C-phycoerythrin from Nostoc sp. was obtained for the first time from limestone drought conditions, showing the need of studying microorganisms from extreme environments.

Next-generation DNA sequencing technology was applied to generate molecular data from semiarid reservoirs during well-defined seasons. Target sequences of 16S-23S rRNA ITS and cpcBA-IGS were used to reveal the taxonomic groups of cyanobacteria present in the samples, and genes coding for cyanotoxins such as microcystins (mcyE), saxitoxins (sxtA), and cylindrospermopsins (cyrJ) were investigated. The presence of saxitoxins in the environmental samples was evaluated using ELISA kit. Taxonomic analyses of high-throughput DNA sequencing data showed the dominance of the genus Microcystis in Mundaú reservoir. Furthermore, it was the most abundant genus in the dry season in Ingazeira reservoir. In the rainy season, 16S-23S rRNA ITS analysis revealed that Cylindrospermopsis raciborskii comprised 46.8% of the cyanobacterial community in Ingazeira reservoir, while the cpcBAIGS region revealed that C. raciborskii (31.8%) was the most abundant taxon followed by Sphaerospermopsis aphanizomenoides (17.3%) and Planktothrix zahidii (16.6%). Despite the presence of other potential toxin-producing genera, the detected sxtA gene belonged to C. raciborskii, while the mcyE gene belonged to Microcystis in both reservoirs. The detected mcyE gene had good correlation with MC content, while the amplification of the sxtA gene was related to the presence of STX. The cyrJ gene was not detected in these samples. Using DNA analyses, our results showed that the cyanobacterial composition of Mundaú reservoir was similar in successive dry seasons, and it varied between seasons in Ingazeira reservoir. In addition, our data suggest that some biases of analysis influenced the cyanobacterial communities seen in the NGS output of Ingazeira reservoir.

Cyanobacterial blooms are increasing worldwide favored by eutrophic conditions of aquatic ecosystems associated with climatic perturbations. Generally, inland lentic systems are more susceptible to the development of harmful blooms. In the Salto Grande Reservoir (Brazil), Microcystis is the most common bloom-forming genus along with a wide range of co-occurring and less-known cyanobacteria taxa. The cyanobacterial community and microcystin production were studied in Salto Grande Reservoir applying biological, toxicological, and molecular approaches. Thirteen cyanobacterial strains belonging to eight genera were isolated and taxonomically investigated based on morphological traits and phylogenetic analyses of their 16S rRNA gene sequence. The morphotypes identified were, in general, in agreement with their phylogeny. The presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) was investigated using PCR gene amplification, which were detected in 76.9 and 84.4% of the strains, respectively. Positive enzyme-linked immunosorbent assays (ELISA) reactions for microcystins were obtained only from the strain Leptolyngbya sp. CENA129. ELISA and high-performance liquid chromatography (HPLC) analyses of the environmental water samples showed the highest microcystin concentration during the dry and rainy seasons, respectively. This study highlights that microcystin production must be suspected in benthic forms as well as in genera that are morphologically similar but belonging to other evolutionary lineages.

Changing global climatic conditions and the continuous eutrophication of aquatic ecosystems have led to increased frequency, duration and toxicity of cyanobacterial blooms worldwide. This makes the provision of time series information on cyanotoxins extremely crucial for effective monitoring and management of water resources. The objective of the present study was to investigate seasonal and annual changes in microcystins (MCs), cylindrospermopsins (CYNs), saxitoxins (STXs), neo-saxitoxin (neo-STX) and anatoxin-a (ATX-a) concentrations in 11 public water supply reservoirs in the semiarid region of Brazil, from 2004 to 2011. Per time, at least one cyanotoxin was present in all the investigated reservoirs. High levels of MCs, CYNs, STXs and Neo-STX were detected simultaneously in Carpina, Duas Unas, Ipojuca, and Jucazinho reservoirs. All the investigated reservoirs had significant concentrations of MCs. The highest levels of MCs were found in Carpina (303,300.0 ng g−1) and Duas Unas (122,053.9 ng g−1) reservoirs, while the lowest concentration of the hepatotoxins was recorded in Ipojuca (10.3 ng g−1) reservoir. On the other hand, CYN was detected in four reservoirs, STXs and Neo-STX in ten reservoirs and ATX-a in two reservoirs. The first record of CYN in Carpina reservoir was obtained in 2006. In addition, Carpina reservoir had the highest concentration of STXs and Neo-STX in the dry and rainy seasons, respectively. The high concentration of cyanotoxins observed in most samples obtained from semiarid reservoirs in Brazil demonstrates the need for regular monitoring and updated management programs.

Cylindrospermopsis raciborskii ITEP-A1 is a saxitoxin-producing cyanobacterium. We report the draft genome sequence of ITEP-A1, which comprised 195 contigs that were assembled with SPAdes and annotated with Rapid Annotation using Subsystem Technology. The identified genome sequence had 3,605,836 bp, 40.1% G+C, and predicted 3,553 coding sequences (including the synthetase genes).

The cyanobacterial population in the Cajati waste stabilization pond system (WSP) from Sao Paulo State, Brazil was assessed by cell isolation and direct microscope counting techniques. Ten strains, belonging to five genera (Synechococcus, Merismopedia, Leptolyngbya, Limnothrix, and Nostoc), were isolated and identified by morphological and molecular analyses. Morphological identification of the isolated strains was congruent with their phylogenetic analyses based on 16S rDNA gene sequences. Six cyanobacterial genera (Synechocystis, Aphanocapsa, Merismopedia, Lyngbya, Phormidium, and Pseudanabaena) were identified by direct microscope inspection. Both techniques were complementary, since, of the six genera identified by direct microscopic inspection, only Merismopedia was isolated, and the four other isolated genera were not detected by direct inspection. Direct microscope counting of preserved cells showed that cyanobacteria were the dominant members (>90%) of the phytoplankton community during both periods evaluated (summer and autumn). ELISA tests specific for hepatotoxic microcystins gave positive results for six strains (Synechococcus CENA108, Merismopedia CENA106, Leptolyngbya CENA103, Leptolyngbya CENA112, Limnothrix CENA109, and Limnothrix CENA110), and for wastewater samples collected from raw influent (3.70 μg microcystins/l) and treated effluent (3.74 μg microcystins/l) in summer. Our findings indicate that toxic cyanobacteria in WSP systems are of concern, since the treated effluent containing cyanotoxins will be discharged into rivers, irrigation channels, estuaries, or reservoirs, and can affect human and animal health.

A cyanobacterial mat colonizing the leaves of Eucalyptus grandis was determined to be responsible for serious damage affecting the growth and development of whole plants under the clonal hybrid nursery conditions. The dominant cyanobacterial species was isolated in BG-11 medium lacking a source of combined nitrogen and identified by cell morphology characters and molecular phylogenetic analysis (16S rRNA gene and cpcBA-IGS sequences). The isolated strain represents a novel species of the genus Brasilonema and is designated Brasilonema octagenarum strain UFV-E1. Thin sections of E. grandis leaves analyzed by light and electron microscopy showed that the B. octagenarum UFV-E1 filaments penetrate into the leaf mesophyll. The depth of infection and the mechanism by which the cyanobacterium invades leaf tissue were not determined. A major consequence of colonization by this cyanobacterium is a reduction in photosynthesis in the host since the cyanobacterial mats decrease the amount of light incident on leaf surfaces. Moreover, the cyanobacteria also interfere with stomatal gas exchange, decreasing CO2 assimilation. To our knowledge, this is the first report of an epiphytic cyanobacterial species causing damage to E. grandis leaves.

Twelve populations of filamentous, heterocytous scytonematoid cyanobacteria from subaerophytic (mainly epiphytic) habitats in subtropical and tropical Brazil (Sao Paulo) were studied. The populations form a uniform cluster, which differs from the traditional scytonematoid genera genetically and by several indistinct, but typical morphological characters (fasciculation of filaments, rare false branching). Two strains were isolated in monospecific cultures. Molecular analyses were performed on these strains from natural populations taken from bromeliad leaves and iron water pipes. Because the results from 16S rRNA gene sequencing indicated the separate position of this cyanobacterium at the generic level, a new genus, Brasilonema, is proposed with the type species Brasilonema bromeliae. The new genus is described using combined molecular and cytomorphological criteria, in accordance with the nomenclatorial recommendations of both the Bacteriological Code and the Botanical Code of Nomenclature (cf. Oren 2004). The ultrastructure of the type species was studied, and vacuole-like structures in the cells were explained. The genus Brasilonema is commonly distributed, particularly in subaerophytic habitats from southeastern Brazil. The type species (B. bromeliae) has a rather curious ecology, living epiphytically (in phytothelmes) inside the vase-shaped leaf rosettes of bromeliads.

Florações de cianobactérias tóxicas em corpos dágua doce usados como fonte para o consumo humano, recreação e irrigação são freqüentes nos dias de hoje devido à eutrofização destes ambientes. O monitoramento de linhagens tóxicas é importante para a prevenção dos efeitos adversos causados por suas toxinas na saúde de humanos e animais. Métodos rápidos e sensíveis para a detecção precoce das cianobactérias tóxicas em estações de tratamento de água e em programas de monitoramento de mananciais são de fundamental interesse para a prevenção desses efeitos. Atualmente, contagem de células, identificação de cianobactérias por microscopia óptica e análises químicas ou imunológicas das toxinas são usadas nos monitoramentos. Em anos recentes, métodos moleculares estão sendo desenvolvidos e propostos para o diagnóstico rápido e sensível da presença de cianobactérias tóxicas em diversos ambientes. Este estudo teve por objetivo implementar uma metodologia capaz de acessar e quantificar as cianobactérias do gênero Microcystis na Praia dos Namorados, no reservatório de Salto Grande, Americana, SP, local cujo uso recreacional é bastante intenso e florações de cianobactérias são freqüentemente observadas. Simultaneamente, buscou-se detectar o potencial de produção de microcistinas desses organismos. A técnica de PCR Quantitativa em Tempo Real (qPCR) foi empregada com essa finalidade e dois conjuntos de oligonucleotídeos LUX foram desenvolvidos tendo como alvo dois genes distintos. Seqüências de cpcBA-IGS do operon da ficocianina (PC) foram obtidas para sete linhagens de cianobactérias brasileiras, as quais foram alinhadas com outras seqüências existentes em banco de dados público, e permitiram o desenho dos iniciadores de qPCR QPCF/QPCR, capazes de amplificar fragmentos de 144 pb. Da mesma forma, outras 11 sequências inéditas foram obtidas para uma região do domínio da N-methyltransferase do gene da sintetase de microcistinas (mcyA) e permitiram o desenvolvimento dos iniciadores QmcyANMTF/ QmcyA-NMTR, capazes de amplificar fragmentos de 154 pb. Ambos os conjuntos foram marcados uma única vez com os fluoróforos FAM (QPCF/QPCR) ou JOE (QmcyA-NMTF/QmcyA-NMTR). Curvas de calibração para ambos os genes foram estabelecidas com regressão linear simples usando os valores de Ct (número de ciclos da qPCR em que a fluorescência atinge um limiar fixo pré-determinado) e concentrações conhecidas de DNA gênomico (em número de células equivalente) da Microcystis sp. NPLJ-4 (produtora de microcistina). As diluições utilizadas para o estabelecimento dessas curvas variaram de 1:10 a 1:10-5 e as concentrações de DNA foram correlacionadas com o respectivo número de células na amostra, obtido pela técnica de Utermöhl em laboratório certificado, como metodologia independente. Para PC e mcyA, as equações de regressão foram y = 43,977 - 1,8097Ln(x) (R2 = 0,99, p

The halogenated compounds aeruginosin and cyanopeptolin (protease inhibitors) are well documented in several cyanobacteria. In this work, the presence of genetic coding for aeruginosin and cyanopeptolin in Sphaerocavum genus and their characterization by mass spectrometry and potential toxic effects were investigated. Three strains of Sphaerocavum brasiliense (CCIBt 3094, CCIBt 3096 and CCIBt 3316) isolated from different reservoirs in Sao Paulo State, and one (CCIBt 3179) from Rio Grande do Norte State were selected for this study. Fragments of aerA-aerB and mcnC-mcnE gene regions (aeruginosin and cyanopeptolin, respectively) were amplified by polymerase chain reaction (PCR) and sequenced. Compounds were characterized by mass spectrometry. All the studied strains presented aeruginosin and cyanopeptolin synthetase genes. Mass spectrometry showed the presence of several aeruginosin and cyanopeptolin variants. The Allium test showed that the ethanolic extract promoted toxic and cytotoxic actions in acute and chronic treatments. These naturally occurring protease inhibitors can play an important role in the physiopathology of human diseases.

While evaluating several laboratory-cultured cyanobacteria strains for the presence of paralytic shellfish poison neurotoxins, the hydrophilic extract of Microcystis aeruginosa strain SPC777--isolated from Billings's reservoir, São Paulo, Brazil--was found to exhibit lethal neurotoxic effect in mouse bioassay. The in vivo test showed symptoms that unambiguously were those produced by PSP. In order to identify the presence of neurotoxins, cells were lyophilized, and the extracts were analyzed by HPLC-FLD and HPLC-MS. HPLC-FLD analysis revealed four main Gonyautoxins: GTX4(47.6%), GTX2(29.5%), GTX1(21.9%), and GTX3(1.0%). HPLC-MS analysis, on other hand, confirmed both epimers, with positive Zwitterions M(+) 395.9 m/z for GTX3/GTX2 and M(+) 411 m/z for GTX4/GTX1 epimers.The hepatotoxins (Microcystins) were also evaluated by ELISA and HPLC-MS analyses. Positive immunoreaction was observed by ELISA assay. Alongside, the HPLC-MS analyses revealed the presence of [L: -ser(7)] MCYST-RR. The N-methyltransferase (NMT) domain of the microcystin synthetase gene mcyA was chosen as the target sequence to detect the presence of the mcy gene cluster. PCR amplification of the NMT domain, using the genomic DNA of the SPC777 strain and the MSF/MSR primer set, resulted in the expected 1,369 bp product. The phylogenetic analyses grouped the NMT sequence with the NMT sequences of other known Microcystis with high bootstrap support. The taxonomical position of M. aeruginosa SPC777 was confirmed by a detailed morphological description and a phylogenetic analysis of 16S rRNA gene sequence. Therefore, co-production of PSP neurotoxins and microcystins by an isolated M. aeruginosa strain is hereby reported for the first time.

Cyanobacterial strains isolated from terrestrial and freshwater habitats in Brazil were evaluated for their antimicrobial and siderophore activities. Metabolites of fifty isolates were extracted from the supernatant culture media and cells using ethyl acetate and methanol, respectively. The extracts of 24 isolates showed antimicrobial activity against several pathogenic bacteria and one yeast. These active extracts were characterized by Q-TOF/MS. The cyanobacterial strains Cylindrospermopsis raciborskii 339-T3, Synechococcus elongatus PCC7942, Microcystis aeruginosa NPCD-1, M. panniformis SCP702 and Fischerella sp. CENA19 provided the most active extracts. The 50 cyanobacterial strains were also screened for the presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes and microcystin production. Putative fragment genes coding for NRPS adenylation domains and PKS keto-synthase domains were successfully PCR amplified from 92% and 80% of cyanobacterial strains, respectively. The potential therapeutical compounds siderophores were detected in five cyanobacterial isolates. Microcystin production was detected by ELISA test in 26% of the isolates. Further a protease inhibitor substance was detected by LC-MS/MS in the M. aeruginosa NPLJ-4 extract and the presence of aeruginosin and cyanopeptolin was confirmed by PCR amplification using specific primers, and sequenced. This screening study showed that Brazilian cyanobacterial isolates are a rich source of natural products with potential for pharmacological and biotechnological applications.

As florações de cianobactérias em reservatórios de águas utilizadas para consumo humano são freqüentes hoje em dia e normalmente são atribuídas à crescente eutrofização destas águas. Em anos recentes o aparecimento de linhagens de cianobactérias produtoras de toxinas tem preocupado bastante os responsáveis pelo monitoramento da qualidade das águas, uma vez que estas toxinas representam risco para a saúde pública. A detecção precoce da presença de linhagens tóxicas nesses reservatórios é essencial para que ações corretivas de controle das florações tenham sucesso. No presente trabalho, a diversidade de cianobactérias presentes em alguns locais das represas Billings e Guarapiranga foi avaliada utilizando a técnica de DGGE (eletroforese em gel de poliacrilamida com gradiente desnaturante) e/ou construções de mini-bibliotecas de amplicons do gene de rRNA 16S e da região do espaço intergênico da ficocianina (PC-IGS). A DGGE, utilizando os iniciadores CYA359F/CYA781R específicos para o gene de rRNA 16S de cianobactérias, mostraram que estes organismos estavam presentes nas onze amostras de água analisadas. Microcystis aeruginosa (94-97% de identidades), Geitlerinema (89% de identidade) e Synechococcus (89% de identidade) puderam ser identificadas. A mini-biblioteca construída com os amplicons de rRNA 16S obtidos usando os iniciadores 27F1/1494Rc produziu seqüências de outro grupo de bactéria (Actinobacteria), indicando a inespecificidade desses iniciadores. Entretanto, na mini-biblioteca construída com os amplicons de PC-IGS obtidos usando os iniciadores PCβF/PCαR, somente seqüências de cianobactérias foram geradas. Nessa mini-biblioteca foram identificadas várias linhagens de Microcystis aeruginosa (98-100% de identidades) e também de Anabaena (89% de identidade). Esses dois gêneros de cianobactérias conhecidos por produzirem microcistinas, foram detectados na amostra de água que continha alta concentração desta toxina (23,49 μg/L). Os oligonucleotídeos iniciadores OMETF/OMETR foram desenhados para amplificar uma região pequena e variável do domínio da N-metiltransferase do gene mcyA e foram testados em treze espécies de cianobactérias isoladas das represas Billings e Guarapiranga. Seqüências geradas por esses iniciadores foram isoladas, clonadas e sequenciadas com sucesso em duas espécies controle (M. aeruginosa NPJB1 e M. panniformis SPC702) e em dois isolados das represas (M. aeruginosa SPC777 e M. protocystis SPC697). A amplificação dessa região do mcyA a partir dos DNAs dos isolados de cianobactérias permitiu identificar linhagens do gênero Microcystis potencialmente produtoras da toxina microcistina. Essa técnica uma vez bem estabelecida para organismos isolados, poderá proporcionar base suficiente para uma melhor detecção de comunidades aquáticas de cianobactérias potencialmente produtoras de microcistinas em ambientes naturais. Cyanobacterial blooms in water reservoirs used for human consumption are frequent nowadays and are usually attributed to the increasing water eutrophization. In recent years, the appearance of toxin-producing cyanobacterial strains has concerned managers of water quality since these toxins represent a public health risk. Early detection of the presence of toxic strains in these reservoirs is essential for the success of bloom control corrective actions. In the present work, cyanobacterial diversity was evaluated in several sites of Billings and Guarapiranga reservoirs using DGGE (denaturing gradient gel eletrophoresis) and/or mini-library constructions of both 16S rRNA gene and phycocyanin intergenic spacer (PC-IGS) region amplicons. The DGGE, using CYA359F/CYA781R primers, specific for cyanobacterial 16S rRNA gene, showed the presence of these organisms in the eleven water samples analyzed. Microcystis aeruginosa (identities of 94-97%), Geitlerinema (identity of 89%) and Synechococcus (identity of 89%) were identified. The mini-library constructed with 16S rRNA amplicons obtained using 27F1/1494Rc primers produced sequences of another group of bacteria (Actinobacteria), indicating the unespecificity of these primers. However, the mini-library constructed with PC-IGS amplicons obtained using PCβF/PCαR primers, generated cyanobacterial sequences only. In this mini-library several Microcystis aeruginosa strains (identities of 98-100%) and Anabaena (identity of 89%) were identified. These two cyanobacterial genera known to produce microcystins were detected in a water sample containing a high concentration of this toxin (23.49 g/L). The OMETF/OMETR primers were designed to amplify a small and variable region of the N-methyltransferase domain of mcyA gene and were tested in thirteen cyanobacterial strains isolated from Billings and Guarapiranga reservoirs. Sequences generated with these primers were successfully isolated, cloned and sequenced in two control species (M. aeruginosa NPJB1 and M. panniformis SPC702) and in two isolates from these reservoirs (M. aeruginosa SPC777 and M. protocystis SPC697). The amplification of this region of mcyA using cultured cyanobacteria DNAs allowed the identification of Microcystis strains with the potential to produce microcystin toxin. This technique, after it is well established for cultured organisms, will provide a basis for better detection of potential microcystin-producing cyanobacterial aquatic communities in natural environments.